Heritable reduction in insect feeding on brassicaceae plants

ABSTRACT

A method for producing plants of the Brassicaceae family that have reduced feeding by cruciferous insects is disclosed. The method comprises selecting for the heritable trait of altered total non-seed glucosinolate levels or for the heritable trait of increased myrosinase activity. Selection may be performed on Brassicaceae cultivars, mutagenized populations or wild populations, including the species Brassica napus, B. campestris and Arabidopsis thaliana. Plants having such altered levels show reduced feeding by cruciferous insects, including flea beetle, diamond back moth and cabbage butterfly. Plants selected for altered levels of both glucosinolates and myrosinase also show reduced feeding by cruciferous insects.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Some of the research disclosed herein was supported by United StatesDepartment of Agriculture Grant Nos. 92-37303-7613 and 93-37302-9572,and National Science Foundation Grant No. BSR-9021451. The United Statesgovernment may have certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to plants having a heritable reduction in feedingby insects upon cotyledons or leaves. More particularly, the inventionrelates to plants of the family Brassicaceae that have a heritablealteration in total non-seed glucosinolate levels and/or alteration inmyrosinase levels that results in a reduction in susceptibility tofeeding damage by cruciferous insects.

BACKGROUND OF THE INVENTION

Plants of the family Brassicaceae comprise species important inagriculture and research. Plants within this family include Brassicarapa (campestris), B. napus, B. oleraceae and Arabidopsis thaliana.Certain members of the genus Brassica are also known as oilseed rape orcanola and are useful in the production of industrial and edible oils,including canola oil. Arabidopsis thaliana is often used for researchpurposes.

Efficient and cost-effective production from such plants is subject tonumerous problems, including damage due to feeding by various insects.Insects that can cause serious damage to Brassicaceae include fleabeetles (e.g., Phyllotreta crucifereae and P. striolata (F.)). Theseinsects are members of the order Coleoptera and cause damage to leavesand cotyledons of Brassicaceae plants in both the larvae and adultstages. Other insects which can cause serious damage to Brassicaceaeplants include members of the order Lepidoptera, such as diamond backmoth (Plutella xylostella) and cabbage butterfly (Pieris rapea).

Glucosinolates are a family of sulfur-containing compounds found in manyplant species, including members of the Brassicaceae. Larsen, P., in TheBiochemistry of Plants, Conn, E., ed., Vol. 7, Academic Press, New York,1981, pp. 501-525; Chew, F., in Chemical Mediation of Coevolution,Academic Press, New York, 1988, pp. 81-112. Glucosinolates contain aglycoside moiety and a variable side chain, including aliphatic alkenylgroups, indolyl groups or aralkyl groups.

Glucosinolates can be hydrolyzed by myrosinase (thioglucosideglucohydrolase, E.C. 3.2.3.1) at the S-glucose bond to produce D-glucoseand an unstable aglycone. The aglycone rearranges to form sulfate andproducts that depend on the particular side chain of the compound beinghydrolyzed. Breakdown products include isothiocyanates (volatile mustardoils), thiocyanates and nitriles (Larsen, supra). Myrosinase isozymesappear to differ in pH optima but do not appear to differ in theirspecificity for particular glucosinolate compounds.

Glucosinolates and myrosinase can occur in all parts of the plant, butare separated cellularly or subcellularly in intact plant tissue. Whenplant tissue is damaged, e.g., by crushing or by insect feeding,myrosinase and glucosinolates are brought into contact and production ofthe hydrolysis products ensues (Chew, F., supra).

Glucosinolate levels in the plant can vary with environmental factorssuch as nutrients (e.g., available soil nitrogen), water, and lightintensity. Louda, S. and Rodman, J., J. Chem. Ecol. 9:397-422 (1983);Wolfson, J., Environ. Entomol. 11:207-213 (1982); Gershenzon, J., RecentAdv. Phytochem. 18:273-320 (1984).

Glucosinolates and glucosinolate degradation products render the mealproduced from rape or canola seeds unpalatable or toxic to animals towhom the meal is fed. During the last two decades, rape varieties havebeen bred that have a seed-specific reduction in glucosinolate levels.It is also known that, in seeds, low glucosinolate levels are recessiveto high glucosinolate levels.

Studies have been performed to ascertain the effect of glucosinolates oninsect behavior. Glucosinolate-related compounds have been painted onplants, and glucosinolates and glucosinolate-related compounds have beenadded to artificial diets, followed by observations of any effects oninsect behavior. The results indicate that such compounds can affectfeeding behavior and oviposition responses (Chew 1988). In general,glucosinolates and their breakdown products may act as ovipositiondeterrents and feeding toxins to non-adapted herbivores, and asattractants and feeding stimulants to species of insect herbivores thatspecialize on mustards. Examples of mustard specialists include fleabeetles, cabbage butterfly and diamondback moth. Of particularimportance is the herbivore P. cruciferae which mainly attacks seedlingsand which is known to have a significant negative impact on total seedset, seed weight, and seedling survivorship of rape plants. (Pilson, D.,Bull. Ecol. Soc. Am. 74:394 (1993).

SUMMARY OF THE INVENTION

Methods are disclosed for producing a plant having a heritable reductionin susceptibility to cotyledon or leaf feeding by cruciferous insects.The method comprises: selecting, in a population of P₀ Brassicaceaeplants, at least one P₀ plant having a total non-seed glucosinolatelevel that is decreased sufficiently, relative to the total non-seedglucosinolate level in the P₀ population, to reduce susceptibility tocotyledon or leaf feeding by cruciferous insects; producing progeny(termed P₁ plants) from the P₀ plant; and identifying at least one P₁plant that inherits the decreased total non-seed glucosinolate level,thereby producing a plant having reduced susceptibility to cotyledon orleaf feeding by insects. The P₀ plant may be selected from plants in the0-15 percentile for total non-seed glucosinolates in the P₀ population.

In another embodiment, at least one P₀ plant is selected from apopulation of P₀ Brassicaceae plants having a mean total non-seedglucosinolate level. The P₀ plant has a total non-seed glucosinolatelevel that is increased sufficiently, relative to the total non-seedglucosinolate level in the P₀ population, to reduce susceptibility tocotyledon or leaf feeding by cruciferous insects. P₁ progeny areproduced for the at least one P₀ plant and at least one P₁ plant isidentified that inherits the increased total non-seed glucosinolatelevels, thereby producing a plant having reduced susceptibility tocotyledon or leaf feeding by insects. The P₀ plant may be selected fromplants in the 85-100 percentile for total non-seed glucosinolates in theP₀ population.

In another embodiment, at least one P₀ plant is selected from apopulation of P₀ Brassicaceae plants having a mean myrosinase activity.The P₀ plant has a level of myrosinase activity that is increasedsufficiently, relative to the myrosinase activity in the P₀ population,to reduce susceptibility to cotyledon or leaf feeding by cruciferousinsects. P₁ progeny are produced from the P₀ plant and at least one P₁plant is identified that inherits the increased myrosinase activity,thereby producing a plant having reduced susceptibility to cotyledon orleaf feeding by insects. The P₀ plant may be selected from plants in the85-100 percentile for myrosinase activity in the P₀ population.

At least one P₀ plant selected for increased myrosinase activity may befurther selected to have a level of total non-seed glucosinolates thatis increased sufficiently, relative to a mean total non-seedglucosinolate level in the P₀ population, to reduce susceptibility tocotyledon or leaf feeding by cruciferous insects.

P₁ progeny may be produced by selfing. P₁ progeny may also be produced,when a plurality of P₀ plants having altered total non-seedglucosinolate level and/or increased myrosinase activity are selected,by making crosses among the plurality of P₀ plants.

The P₀ population may comprise plants grown from mutagenized seeds. Insome embodiments, a P₀ population may comprise plants geneticallyengineered for altered total non-seed glucosinolate levels. In someembodiments, a P₀ population may comprise plants genetically engineeredfor increased myrosinase activity, e.g., plants having a recombinant DNAconstruct expressing a myrosinase coding sequence.

A P₀ plant or a P₁ plant may be identified by genetic linkage betweenaltered total non-seed glucosinolate level and a polymorphic geneticmarker, e.g., a nucleic acid having substantial sequence similarity toat least 50 nucleotides from Arabidopsis RFLP probe g6842 or pCITd23. Insome embodiments, a P₀ or P₁ plant may be identified by genetic linkagebetween increased myrosinase activity and a polymorphic genetic marker,e.g., a nucleic acid having substantial sequence similarity to at least50 nucleotides from Arabidopsis RFLP probe m106.

A P₁ plant preferably is a Brassica campestris plant or a Brassica napusplant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a histogram showing the frequency distribution of thedevelopmentally adjusted total non-seed glucosinolate levels in a P₀population of Brassica rapa.

FIG. 2 is a histogram showing the frequency distribution ofdevelopmentally adjusted total non-seed glucosinolate levels in a P₂ B.rapa population after selection for decreased glucosinolate levels.

FIG. 3 is a histogram showing the frequency distribution ofdevelopmentally adjusted total non-seed glucosinolate levels in a P₂ B.rapa population after selection for increased glucosinolate levels.

FIG. 4 is a histogram showing the frequency distribution ofdevelopmentally adjusted myrosinase activity in a P₀ B. rapa population.

FIG. 5 is a histogram showing the frequency distribution ofdevelopmentally adjusted myrosinase activity in two P₂ B. rapapopulations selected for decreased (upper histogram) or increased (lowerhistogram) myrosinase activity.

FIG. 6 is a bar graph comparing the fitness, herbivory and myrosinaseactivity for the two P₂ B. rapa populations of FIG. 5. The fitness,herbivory and myrosinase activity values for the increasedmyrosinase-selected population were each set at 100 and thecorresponding values for the decreased myrosinase-selected populationdetermined.

FIG. 7 is a response surface for diamondback moth herbivory, totalnon-seed glucosinolate levels and myrosinase activity for 39 Arabidopsisthaliana homozygous recombinant inbred lines.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Heretofore, it has not been possible to predict whether heritablealterations in glucosinolate levels can be selected in Brassicaceaeplant populations or, if such alterations could have been selected,whether insect feeding on whole plants would be affected. The inventorshave discovered that a plant of the family Brassicaceae can be bred tohave a heritable alteration in the level of total non-seedglucosinolates or the level of myrosinase activity. Surprisingly, plantshaving such heritable traits can be selected and show a reduction insusceptibility to cotyledon and leaf feeding by insect pests, includingflea beetles and diamondback moths.

A plant according to the invention is produced from a population ofBrassicaceae plants from a single species, herein termed a P₀population. This population may be, for example, a single uniformpopulation of wild plants, an F₁ population derived from two cultivars,or a population of plants from a single cultivar. The size of the P₀population to be used depends upon the genetic variation present in thepopulation and on the intensity of the selection pressure that is to beapplied. See, e.g., Downey, R. and Rakow, G. in Principles of CultivarDevelopment, Vol. 2: Crop Species, Fehr, W., Ed., McMillan Publishing,New York, (1987), pages 437-486. Generally, a smaller P₀ population canbe used if a large amount of genetic variation is known or expected tobe present in the population. A smaller P₀ population size, e.g., fromabout 100 to about 1000 plants, also can be used if it is desired toapply less intense selection pressure. On the other hand, a largerpopulation size, e.g., from about 500 to about 30,000 plants, can beused if less genetic variation is present or if more intense selectionpressure is to be applied.

A P₀ population may comprise plants grown from mutagenized seeds, whichhave greater genetic variation than corresponding non-mutagenized seeds.Mutagenesis typically increases the genetic variation for total non-seedglucosinolate levels. Chemical mutagens, including but not limited to,ethidium bromide, nitrosoguanidine, diepoxybutane, ethylnitrosourea andethyl methane sulfonate may be used to create a P₀ population.Alternatively, physical mutagens such as X-rays, UV rays may be used.Mutagenesis treatment may also be applied to other plant tissues, suchas cell cultures, embryos, microspores and shoot apices. Plants formedfrom such mutagenized tissues may be used in a P₀ population.

A P₀ population may comprise plants that have been geneticallyengineered for decreased total non-seed glucosinolate levels. Suchgenetic engineering may comprise, for example, sense or anti-senseconstructs designed to alter expression of glucosinolate biosynthesisgenes. Alternatively, such genetically engineered plants may compriseinsertional mutants that eliminate expression of a biosynthesis gene.

Individual plants, e.g., seedlings, in the population are assayed forthe level of total non-seed glucosinolates. Tissues to be analyzed fortotal non-seed glucosinolate levels may be any non-seed tissue, e.g.,stems, cotyledons, leaves and the like. Preferred tissues are cotyledonsor first true leaves of seedlings about 6 to about 50 days of age. Theassay is preferably carried out before onset of flowering, so that onlythose plants having the desired level of glucosinolates need topollinated and advanced to the next generation.

Glucosinolate assay methods are known in art. See, e.g., Heaney , R. andFenwick, G., Z. Pflazenzuchtg. 87:89-95 (1981). Assays may measureglucosinolates by, e.g., high performance liquid chromatography (HPLC)analysis of non-seed extracts or the amount of glucose released afterenzymatic hydrolysis of glucosinolates in non-seed extracts. If desired,an assay may be adapted to the use of microtiter plates for rapidanalysis of large numbers of samples. The absolute level of totalnon-seed glucosinolates need not be measured. Rather, measuring thelevel relative to the average of the population is sufficient to performthe method of the invention.

From the glucosinolate analysis a frequency distribution of totalnon-seed glucosinolate levels may be plotted and individual plantsselected for advancement to the next generation. A plant producedaccording to the invention may be selected to have either a higher or alower level of total non-seed glucosinolates than the starting P₀population from which such a plant is derived. Those plants having atotal glucosinolate level about 1/2 standard deviation above or belowthe mean glucosinolate level of the P₀ population are likely candidatesto have a heritable alteration in total glucosinolate level. Such plantswill typically be in either the 0-15 percentile or the 85-100 percentilefor total non-seed glucosinolate level.

If desired, P₀ plants may be evaluated on the basis of a Z-distribution.Zar, J., Biostatistical Analysis, 2nd Ed., Prentice-Hall, EnglewoodCliffs, N.J., 1984, pp. 83-86. Plants that exceed the upper or lowerstatistical thresholds are advanced to the next generation. Thestatistical threshold chosen will depend upon the population size, thegenetic variation known or expected in the population and the desiredselection intensity, as is known in the art.

Although a selected plant possesses the heritable trait of alterednon-seed glucosinolates, absolute glucosinolate levels will varydepending on growing conditions to some extent. Nevertheless, comparedto the starting P₀ population, a significant difference in non-seedglucosinolate levels is observed between the P₀ population and a plantproduced according to the invention.

One or more selected P₀ plants are selfed, or crossed to another P₀plant having altered total glucosinolate levels. The choice betweenselfing or crossing to another P₀ plant will to some extent bedetermined by the species of plants. P₀ plants preferably are selfedwhen using a B. napus population or certain B. campestris cultivars. Asan alternative, a plurality of P₀ plants may be identified and suchplants may be intermated, i.e., crosses made among the plurality of P₀plants having the same directional alteration in total non-seedglucosinolates.

P₁ seeds are replanted and assayed for total non-seed glucosinolatelevels as described above and P₁ plants having the desired alteredglucosinolate level are identified. The statistical threshold forselection of P₁ plants typically is as stringent as, or less stringentthan, the threshold used in the initial selection.

Progeny of the P₁ plant or plants typically undergo further breeding,e.g., selfs, backcrosses, or pedigree selection, to yield a line orcultivar. Breeding to produce a line or cultivar incorporates desiredcharacteristics such as yield, standability, disease resistance andmaturity as well as the desired heritable level of total non-seedglucosinolates. Typically, progeny of P₁ plants (P₂ seed) are grown outand at least one P₂ plant inheriting an altered total glucosinolatelevel is selected as described above. The breeding and selection processis continued in subsequent generations in order to produce a line orcultivar having the trait of heritable reduction in total non-seedglucosinolate levels.

If desired, one or more P₂ plants may be outcrossed to a population ofBrassicaceae plants different from the P₀ population, e.g., a differentcultivar, in order to transfer the trait into other cultivars or otherspecies. One or more P₂ plant may also be outcrossed to plants of adifferent Brassicaceae species, e.g., a selected B. campestris plant maybe crossed to B. napus. In addition, backcross or pedigree selectionmethods may be used in order to confer additional desirable agronomictraits into a line.

In an embodiment of the invention, a polymorphic genetic marker that isgenetically linked to altered total non-seed glucosinolate levels may beused to identify or select a plant to be used in subsequent crosses. Theuse of such markers, often termed "marker-assisted selection",facilitates the selection process by increasing the likelihood that theobserved glucosinolate phenotype is due to the underlying heritablegenetic trait. Types of markers that are suitable for marker-assistedselection are known in the art and include, without limitation, genomicprobes such restriction fragment length polymorphisms (RFLPs) orPCR-based polymorphisms, single-stranded conformational markers (SSCPs),denaturing gradient gel electrophoresis markers (DGGEs), randomamplification polymorphism DNA (RAPDS) and microsatellite markers.Aguade, M., W. Myers, A. D. Long, and C. H. Langley, Proc. Natl. Acad.Sci. USA 91:4658-4662 (1994); Rafalski, J. A., and S. V. Tingey, Trendsin Genet. 8:275-280 (1993); Lessa, E. P. and G. Applebaum, Molec. Ecol.2:119-129 (1993); Konieczny, A. and F. Ausubel, Plant J. 4:403-410(1993).

An illustrative example of a suitable polymorphic marker for selectionin Brassicaceae is cosmid g6842, which carries an Arabidopsis genomicDNA fragment from chromosome 2. Lister, C. and Dean, C., Plant J.4:745-750 (1993). Another useful polymorphic marker is contained incosmid pCITd23, which is located on chromosome 4. Lister, C. and Dean,C., supra. Other polymorphisms may be present in cosmids g6842 orpCITd23 which are useful for marker-assisted selection in populationsother than A. thaliana. Furthermore, polymorphisms located outside ofthe polymorphisms present in g6842 or pCITd23, but genetically linked tog6842 or pCITd23 polymorphisms are suitable for use in the invention. Ingeneral, polymorphic markers useful in the invention are within about 25centiMorgans (cM) of the altered total glucosinolate trait, preferablywithin about 15 cM, more preferably within about 5 cM of the trait.

Other useful markers include nucleic acids having substantial sequencesimilarity to the g6842 or pCITd23 fragments. Such nucleic acidstypically are substantially similar to about 50 nucleotides or more of apolymorphic sequence in g6842 or pCITd23. Nucleic acids havingsubstantial sequence similarity may be identified by their ability tohybridize to probes in g6842 or pCITd23. Hybridization to identifynucleic acids having substantial sequence similarity may be carried outunder stringent conditions as described in widely recognized protocols.See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual (2ndEdition), Cold Spring Harbor Laboratory Press (1989), pp. 1.101-1.104;9.47-9.58 and 11.45-11.57. Generally, high stringency conditions reflectat least one wash of the hybridization membrane in 0.05× to 0.5×SSC with0.1% SDS at 65° C., or washing conditions of equivalent stringency.

Markers polymorphic in a first population may not be polymorphic in asecond, different Brassicaceae population. Methods are known in the artfor identifying polymorphisms useful in the second population based ontheir association with a polymorphism useful in the first population.For example, genomic DNA from the second population can be digested withdifferent restriction enzymes than those used with the first population.A PCR-based polymorphic marker from the first population may be clonedand the cloned fragment tested as an RFLP probe with the secondpopulation. Another procedure for obtaining polymorphisms uses bulkedsegregant analysis to identify a one or more additional RAPD markerslinked to the first RAPD marker. The additional RAPD markers can betested directly in the second population or can be cloned and the clonedfragment tested as an RFLP or PCR-based marker in the second population.Michelmore, R. et al., Proc. Natl. Acad. Sci. USA 88:9828-9832 (1991).

In another embodiment of the invention, a population of P₀ Brassicaceaeplants is assayed for an increased level of myrosinase enzyme activityin non-seed tissues, preferably cotyledon or leaf tissues. A suitable P₀population comprises plants as described above for total non-seedglucosinolate selection. For example, a P₀ population may compriseplants derived from mutagenized seeds. Alternatively, a P₀ populationmay comprise plants genetically engineered for increased myrosinaseactivity, e.g., having a recombinant DNA construct expressing amyrosinase coding sequence. Chadchawan, S., et al., Plant Phys.105:671-672 (1993). Such constructs typically include regulatorysequences such as a promoter and enhancer sequences for high levelexpression, preferably in non-seed tissues.

Selection for increased levels of myrosinase is carried out in a mannersimilar to that described above for glucosinolate levels, e.g., at theP₀, P₁, and, optionally, P₂ and subsequent generations. Assays formyrosinase levels may be carried out by methods known in the art, forexample, removing endogenous glucosinolates and measuring myrosinaseenzyme activity spectrophotometrically. As discussed above forglucosinolate assays, myrosinase assays preferably are carried out andcompleted before flower emergence, so that a plant or plants having thedesired myrosinase activity level are identified before pollinations aremade. Statistical thresholds for selecting plants having increasedmyrosinase activity are determined based on the population size, geneticvariation in the population and selection intensity that is applied, asdiscussed above.

Plants developed by a method of the invention may be any species of thefamily Brassicaceae. Preferred species include the spring and winterforms of Brassica campestris and Brassica napus.

In another embodiment of the invention, a population of P₀ Brassicaceaeplants are used to select for a plant carrying both the trait of alteredtotal non-seed glucosinolate level and the trait of increased myrosinaseactivity.

The inventors have found that plants having heritable alterations intotal non-seed glucosinolate levels show significantly reducedsusceptibility to leaf feeding by adult and larval forms of cruciferousinsect pests of Brassicaceae. Cruciferous insects include flea beetles,diamond back moths and cabbage butterflies. Leaf damage may be reducedby more than 10% compared to control plants. Similarly, populationsinheriting an increased level of myrosinase, or increased levels of bothtotal non-seed glucosinolates and myrosinase activity, show decreasedsusceptibility to leaf damage by coleopteran insect pests of Brassica.Such damage may be reduced by more than 10% compared to control plants.

Plants of the invention are advantageous in that alterations inglucosinolate levels or myrosinase levels, or both, reduce insect damagecompared to plants of the P₀ population. Many cruciferous insects,including diamond back moths, flea beetles and cabbage butterfliesprefer to feed on young seedlings, resulting in yield losses andrequiring the use of insecticides to control insect damage. Plantsproduced according to the invention can respond more vigorously toinsect attack, resulting in higher plant counts in the field andincreased yield relative to P₀ plants. Moreover, such plants can reducethe use of insecticides, thereby lowering the cost of production forfarmers.

Plants produced according to the invention are advantageous in that suchplants may now be grown in areas where high insect populations precludethe use of rape, canola, or turnip rape as a crop. Moreover, a reductionin insect leaf or cotyledon feeding leads to less physiological stresson plants, which results in more consistent seed quality traits, such asoil and protein produced from such seeds. Plants according to theinvention are more likely to mature at the expected time, resulting inless chlorophyll in seeds, and to higher oil quality.

Because genetic control of glucosinolate levels in cotyledons and leavesis separable from levels in seeds, insect damage to non-seed tissues canbe reduced by methods of the invention while maintaining lowglucosinolate levels in canola quality rape seeds.

The invention will be further understood with reference to the followingillustrative embodiments, which are purely exemplary, and should not betaken as limiting the true scope of the present invention as describedin the claims.

EXAMPLES Example 1 Selection for altered glucosinolate levels

Seeds from a wild population of B. rapa were collected in the BitterrootValley, Montana. Seeds from 45 plants in the wild population werecollected and pooled. This bulk seed collection was used to select forhigh or low total non-seed glucosinolate concentrations. The selectionexperiment was started with 500 seedlings, which population size issufficient to minimize genetic drift and inbreeding effects.

B. rapa is an outcrossing species, which minimizes the effects oflinkage disequilibrium. Crow, J. and Kimura, M., An Introduction topopulation Genetics Theory, Burgess Publishing, Minneapolis, Minn.,Chapter 2. Linkage disequilibrium refers to statistical dependencebetween genotype frequencies at two loci and can cause genotypes to havecorrelated values for two traits even when there is no causal orfunctional relationship between these traits. Linkage disequilibrium canconfound the effects of many loci, particularly in wide crosses, e.g.,interspecific crosses. Young, N. and Tanksley, S., Theor. Appl. Genet.77:353-359 (1989); Giamoustaris, A. and Mithen, R., Ann. Appl. Biol.126:347-363 (1995). Consequently, results from studies involving widecrosses can be difficult to interpret.

Total glucosinolate levels in cotyledons were correlated with totalglucosinolate concentrations in true leaves (glucosinolates: r=0.37, 104plants, P=0.001; myrosinase: r=0.72, 104 plants, P<0.001). Selection wasconducted on cotyledons because cotyledons were more easily handled forthe hundreds of assays required in the selection experiments.

Total non-seed glucosinolates were measured by a protocol similar tothat described by Heaney, R. and Fenwick, G., Z. Pflanzenzuchtung87:89-95 (1981). Mini-columns were prepared from 1.2 ml polypropylenetubes by making a small hole in the bottom of each tube with a heatedinoculating needle. The hole was made by forcing the needle from theinside out to promote proper drainage. A small amount of glass wool wasthen packed into the bottom of each column. DEAE Sephadex®-A25(Pharmacia) was hydrated overnight in excess distilled H₂ O (dH₂ O). Theexcess H₂ O was decanted from the Sephadex® slurry and, using adisposable pipet with an enlarged tip, 400 μl of slurry was dispensedinto each column and allowed to drain. Each column had a bed volume of200 μl.

Each column was washed once with 1 ml of 0.5M pyridine acetate (19.8 mlpyridine+15 ml glacial acetic acid, brought to 500 ml with dH₂ O) andtwice with 1 ml of dH₂ O, allowing the column to drain between eachaliquot. If not used immediately, columns were stored at 40° C. withcolumn tips immersed in a small amount of dH₂ O.

One cotyledon was removed from a 7 day old plant and the cotyledon wasscanned under a desktop computer scanner (Hewlett-Packard) linked to apersonal computer. Cotyledon area was determined by a computer programthat counted pixels in the scan; the data were stored in a computerfile. Each cotyledon was placed in a 1.2 ml tube containing 0.5 ml dH2Oand four 3/32 inch ball bearings. Tubes were placed in a 96 well formatrack having holes in the bottom and sides of the rack. The rack andtubes were placed in a glycerol bath and incubated at 100°-1050° C. for5 minutes. No more than 64 tubes were heated at one time, avoiding theuse of wells at the center of the rack. The rack and tubes were thenplaced in ice water to cool. After cooling, the tubes were transferredto a centrifuge rack, capped securely and agitated on a paint shaker forat least 45 seconds to macerate the cotyledon. A lead acetate-bariumacetate solution (0.5 ml, 0.3M each lead acetate and barium acetate in0.29% v/v glacial acetic acid in dH₂ O) was added to the maceratedextract and the tubes were mixed. The extract was then centrifuged at3200 rpm for 10 minutes.

An aliquot of the extract (250 μl) was passed over a charged DEAESephadex®-A25 column. Up to about 500 μl of extract can be used, ifdesired. The column was washed twice with 200 μl of 4M acetic acid, thenwashed three times with 500 μl of dH2O, draining and discarding theeluate after each wash. The rate of elution can be increased by blottingthe tip of the column on a pad of paper towels.

One hundred μl of myrosinase (0.645 U/ml, 3 mg/ml, from Sinapis alba,Sigma Chemical Co.) were added to each column. Columns were covered andincubated overnight at room temperature. Following the myrosinaseincubation, each column was washed with 750 μl dH₂ O and the eluate,containing the glucose reaction product, was collected in a 1.2 ml tube.

An equal volume (200 μl) of each eluate was transferred to a well of amicrotiter plate and 100 μl of glucose oxidase/peroxidase color reagentwas added to each well. Glucose oxidase/peroxidase color reagentcontained equal volumes of reagent #1 and reagent #2. Reagent #1contained glucose oxidase (13 U/ml), 0.8 mM 4-aminoantipyrine andimidazole buffer, pH 7.0. Reagent #2 contained 2.5 U/ml peroxidase, 8.8mM phenol and imidazole buffer, pH 7.0. Imidazole buffer contained0.136M imidazole, pH 7.0, 0.03% w/v sodium azide and 0.42% v/v glacialacetic acid. The microtiter plates were covered and incubated at roomtemperature for one hour. A standard curve was prepared from wellscontaining only glucose and wells containing only reagents. After onehour, the absorbance at 490 nm (A₄₉₀) was measured and the data wasstored in a computer file.

The A₄₉₀ for each cotyledon was divided by the cotyledon area to obtaina specific glucosinolate level (A₄₉₀ per mm² leaf or cotyledon).Specific glucosinolate and myrosinase concentrations decrease asseedlings develop (e.g., in the field, specific glucosinolateconcentration=4.6-61.6 area+250.8 area² ; R² =63%; F=50.9; df=2, 60;P<0.001). To correct for variation in seedling development within apopulation, a developmentally adjusted glucosinolate value wascalculated as the residuals from the regression of specificconcentration on leaf (or cotyledon) area.

The frequency distribution of developmentally adjusted glucosinolateconcentration is shown for the base P₀ population in FIG. 1. Aftereliminating 5% of individuals at each of the low and high extremes toavoid outliers, the 48 highest and 48 lowest individuals were selectedand transplanted to a growth room. Plants within each selected groupwere mass pollinated (random mating within the selected population) toproduce P₁ progeny. For the next generation of selection, at least fourP₁ seeds from each high glucosinolate selected P₀ plant were plantedtogether in a random design and total non-seed glucosinolate levelsdetermined as described above. At least four P₁ seeds from each lowglucosinolate selected P₀ plant were planted and assayed in the samemanner. At least 250 P₁ plants were grown per treatment. Aftercalculating the developmentally adjusted glucosinolate level asdescribed above, 5% tails from the frequency distribution were excludedand the 48 highest and 48 lowest plants from the remaining populationwere selected for advancement to the next generation. Plants within eachgroup were intermated as described above to obtain P₂ progeny.

Variance effective population size was approximately 72 (=1.5×48), dueto uniform numbers of progeny through the maternal component of fitness(Crow, J. and Kimura, M., supra, p. 358). Therefore, genetic drift wasunlikely to be an important factor in these selection experiments.

A histogram of developmentally adjusted glucosinolate levels incotyledons is shown for the low glucosinolate-selected P₂ population inFIG. 2 and for the high glucosinolate-selected P₂ population in FIG. 3.The results indicated that there were statistically significantdifferences between high and low populations after only two generationsof selection. ANOVA results were: r² =6.0%; F=11.36; df=1,3; P=0.043.High and low populations differed from each other by about 1/2 standarddeviation. Statistically significant heritabilities were calculated fromparent-offspring regressions (h² =2 slope! for half sibs; ANOVA: h²=0.17; F=4.75; df=1,208, P=0.03). Falconer, D., Introduction toQuantitative Genetics, 3rd ed., Wiley, N.Y., 1989. These resultsindicate that increased and decreased levels of total non-seedglucosinolate are heritable traits that can be selected for inBrassicaceae plants.

A field experiment with the two glucosinolate-selected populations wasconducted near Corvallis, Oregon, about 16 km from the site where the P₀seeds were originally collected. P₂ seeds from high and low populationswere planted in a randomized manner within each of 10 blocks. Each blockcontained a total of 96 seeds, 48 seeds from each selection treatment.Each block also contained two border rows of non-experimental plants tocontrol for edge effects. Seeds were planted 4 cm apart to control forcompetitive effects. Seeds planted in the field were first germinated inthe laboratory. If seeds from selected plants did not germinate, or ifselected plants produced too few seeds for adequate replication, theirposition in the blocks were filled with non-experimental seed to keepcompetitive effects uniform. Lack of germination or seed shortagesreduced the number of experimental seeds by approximately 20 seeds perblock. These effects were approximately equal between high and lowselected lines (P>>0.05). There were approximately 760 P₂ seeds plantedin the experiment.

A whole block factor of nitrogen fertilizer was added to investigate apotential genotype-by-environment interaction. Fertilizer was added tohalf the blocks at random in each experiment. Before planting, a mixedsampling of soil nitrates from the field showed nitrate levels to be65.23 g/m². Urea (46% N) was added at 4.68 g per 0.26 m² block justbefore planting, which increased soil nitrogen levels to 195.00 g/m².Increasing soil nitrogen levels increases foliage nitrogen levels in B.rapa at this site. Jackson, G., et al., Montana AgResearch 10:21-24(1993).

Thus, in the field, blocks were nested within nitrogen levels and highand low selection treatments were randomized within each block. Thisconstitutes a split plot design, with nitrogen treatmentcross-classified with selection treatment.

Herbivory by flea beetle adults was assessed by counting the number ofuniform pits chewed per leaf on cotyledons and on the first true leaf.Pit counts were divided by leaf size (width for cotyledons, length fortrue leaves) to obtain a damage estimate per cotyledon or leaf area foreach plant. Cotyledon width was also used as a covariate in statisticalanalyses to further control for developmental changes in metaboliteconcentrations.

Herbivory by specialist lepidopteran larvae (diamondback moth andcabbage butterfly) was estimated on a visual scale from 0 to 5 on adultplants at the time most plants began flowering, with 0 representing noinsect damage and 5 representing maximum insect damage. Three observerswere used to estimated herbivory. Only one observer estimated herbivorywithin a particular block, to avoid within-block observer bias.

When fruits were fully developed, the number of fruits and average fruitlength from three fruits that were four, five, and six nodes down on themain inflorescence were recorded. The number of pods multiplied by theaverage pod length was used as an estimate of seed production per plant.Estimated seed production was taken as an estimate of the femalecomponent of fitness for each plant.

Data were analyzed with a computer statistics program (SYSTAT, Evanston,Ill.). Response variables were log transformed before analysis if neededto comply with assumptions of normality. Residuals were examined forhomoscedasticity.

The herbivory data are summarized in Tables 1 and 2. The differencebetween genotypes in each environment was statistically significant(P=0.039).

                  TABLE 1                                                         ______________________________________                                        Herbivory by flea beetles                                                     on glucosinolate populations                                                  Selected       Mean Pit                                                       Population     Number     P value                                             ______________________________________                                        High Gluc.     0.95 +/- 0.036                                                                           0.040                                               Low Gluc.      0.86 +/- 0.037                                                 ______________________________________                                         .sup.a Blocks with added soil nitrogen.                                  

                  TABLE 2                                                         ______________________________________                                        Herbivory by Lepidopteran larvae                                              on glucosinolate populations                                                  Selected     Mean                                                             Population   Score                                                            ______________________________________                                        High         1.29 +/- 0.11                                                    Low          1.36 +/- 0.11                                                    High + N.sup.a                                                                             1.56 +/- 0.10                                                    Low + N.sup.a                                                                              1.34 +/- 0.12                                                    ______________________________________                                         .sup.a Blocks with added soil nitrogen.                                  

Statistically significant differences in levels of flea beetle herbivorywere found between the high and low glucosinolate populations (13%difference in leaf area consumed, p=0.01 by ANOVA). In the absence ofadded soil nitrogen, the high glucosinolate population showed a 9.8%higher levels of herbivory (Table 3). This difference was statisticallysignificant in cotyledons (P=0.040). In true leaves there was asignificant effect of nitrogen (P=0.026; Table 4); plants in blocks withadded nitrogen had 28.6% lower levels of herbivory.

For lepidopteran larvae there was a statistically significantinteraction between nitrogen and glucosinolate selection treatment inthe glucosinolate field experiment (P=0.039; Table 4). In blocks withadded nitrogen, the population selected for low glucosinolateconcentrations had 16% lower herbivory, whereas in the absence of addedsoil nitrogen, the low glucosinolate population had 5% higher herbivory.These results indicate a significant genotype-by-environment interaction(GxE).

Although populations having low total glucosinolate concentrationsshowed reduced leaf and cotyledon feeding by flea beetles, there was nosignificant difference in fitness between low and high selectiontreatments (P=0.056; Table 4).

                  TABLE 3                                                         ______________________________________                                                       Glucosinolate                                                  Population     Concentration                                                                            Herbivory                                           ______________________________________                                        High Glucosinolate                                                                           100.0 +/- 3.0                                                                            100.0 +/- 3.8                                       Low Glucosinolate                                                                             84.2 +/- 2.9                                                                             90.2 +/- 3.8                                       ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        ANOVA tables from split-plot field experiment for high and                    low glucosinolate-selected populations were compared for                      resistance and fitness parameters. Block {N} is the                           variation among blocks nested within the nitrogen (N)                         factor; Selection (S) is the variation between high and low                   selected populations; size is the covariate, cotyledon                        width.                                                                        F ratios.sup.a                                                                                 Cotyledon                                                                              Leaf   Leaf                                                          resistance                                                                             resistance                                                                           resistance                                                    to flea  to flea                                                                              to Lep.                                      Source   df      beetles  beetles                                                                              larvae Fitness                               ______________________________________                                        Nitrogen 1       0.14     7.45*  0.28   0.93                                  Block {N}                                                                              8       18.68*** 10.64***                                                                             4.38***                                                                              18.54***                              Selection (S)                                                                          1       6.00**   0.13   1.67   5.00                                  N*S      1       0.26     0.52   6.10*  0.00                                  S*Block {N}                                                                            8       1.09     1.23   0.29   1.648                                 size     1       0.93     6.69** 4.48*  159.19***                             ERROR     476.sup.s                                                                            0.19     0.03   1.59   0.438                                 ______________________________________                                         .sup.a Mean square (MS) for F ratios and corresponding degrees of freedom     (df) in splitplot design as specified in Steel, J. and Torrie, J.,            Principles and Procedures of Statistics: A Biometrical Approach, 2nd ed.,     McGrawHill, New York (1980).                                                  .sup.s df for MS ERROR ranged from 476 to 544                                 *P<0.05; **P<0.01; ***P<0.001                                            

Example 2 Selection for altered myrosinase levels

Seeds from the base population of Example 1 were used to select forplants having altered levels of myrosinase. A pool of 500 P₀ seeds wasplanted in the greenhouse and a cotyledon was removed from each plantfor myrosinase assays. In the myrosinase assay, endogenous glucose andglucosinolates were removed and myrosinase collected through a desaltingG-25 or G-50 Sephadex® column. An exogenous glucosinolate was added(sinigrin) and the resulting rate of glucose production was measuredspectrophotometrically.

Excess dH₂ O was decanted from Sephadex® G25 or G50 that had beenhydrated overnight, using an Eppendorf repeater pipet equipped with asyringe with an enlarged tip opening. About 300 μl of Sephadex slurrywas transferred to each well of a Silent Monitor™ 96 well membrane testplate (Product NO. SM300LP, Pall Corporation) to form a Sephadex®mini-column. These plates contain 3 micron Loprodyne® membrane bottomedwells. Only wells 1-80 of each plate were used for extracts; wells 81-96were reserved for controls and a glucose standard curve. The preparedplates can be prepared one day before use, covered with Parafilm®, andstored at 40° C. Immediately before use, all excess dH2O was removedfrom the mini-column plates by placing the plate over an empty 96 wellmicrotiter plate and centrifuging in a Hermle Z320 centrifuge at 1000rpm.

An extract from each cotyledon was prepared by placing the cotyledon ina 1.2 ml tube containing four 3/32 inch ball bearings and adding 750 μlof 200 mM Tris HCL, pH 7.5. In some experiments, extraction buffer alsocontained 10 mM EDTA and 2 mM DTT. Tubes were place in a 96 well rackhaving holes in the bottom and sides of the rack. The tubes were cappedsecurely and agitated on a paint shaker for at least 45 seconds tomacerate the cotyledon. One hundred μl of macerated extract weretransferred to a mini-column well. Each extract and Sephadex® mixturewas stirred gently with a pipet tip. The mixture was allowed toequilibrate for 5 minutes at room temperature.

The mini-column plate then was placed over an empty 96 well microtiterplate and the two plates were taped together and centrifuged in aclinical centrifuge. To collect the eluate in the microtiter plate, thecentrifuge was allowed to reach 600 rpm and then immediately shut off.The tape was removed and the eluate-containing microtiter plate was setaside. The mini-column plate was washed with 50 μl of dH₂ O per well forG-25 columns or with 25 μl of dH₂ O for G-50 columns. The mini-columnswere not stirred during the dH₂ O wash. After 5 minutes, the mini-columnplate and the microtiter plate were taped together in the sameconfiguration and centrifuged as described above to collect the secondeluate. The volume of the combined eluates was about 100 μl. It isimportant to centrifuge no faster than 600 rpm, since centrifugation athigher speeds causes the column packing material to become "dry" andincreases the amount of background glucose in the eluate.

Serial dilutions of a glucose standard (1 mM to 0.0625 mM glucose, 100μl per well) were added to wells 81-85 and 89-93. Reagent blanks (100μl) were added to wells 86-88 and 94-96. Positive control microtiterplates contained serial 2-fold dilutions of commercial myrosinase (100μl per well; 2 mg/ml, 0.43 U/ml at the highest concentration) instead ofsample eluate. Negative control plates were prepared by adding 25 μl ofsodium phosphate (33 mM, pH 6.5) to sample wells instead of the sinigrinsubstrate.

The myrosinase reaction was started by adding 25 μl of sinigrin (5 mM,Sigma) to each well of experimental sample plates and positive controlplates. Equal volumes of color reagents #1 and #2 from Example 1 weremixed and 50 μl of the mixture was added to each well. The microtiterplate was covered with Parafilm® and incubated at room temperature for60 minutes. The Parafilm® was then removed, each well was mixed for 10seconds and the A₄₉₀ measured in a BioRad Microplate Reader. The datawas stored in a computer file.

The A₄₉₀ for each cotyledon was divided by the cotyledon area to obtaina specific myrosinase activity (A₄₉₀ per mm² leaf or cotyledon). Adevelopmentally adjusted myrosinase activity was calculated in the samemanner as described for glucosinolates in Example 1. The frequencydistribution of developmentally adjusted myrosinase activity is shownfor the base P₀ population in FIG. 4.

After eliminating 5% of individuals at each of the low and high extremesto avoid outliers, the 48 highest and 48 lowest individuals from the P₀population were selected, transplanted and randomly mated withinselected groups as described in Example 1. After harvest, at least fourP₁ seeds from each high and low myrosinase-selected P₀ plant wereplanted together in a random design and myrosinase activity determinedas described above. At least 250 P₁ plants were grown per treatment.After calculating a developmentally adjusted myrosinase activity in thesame manner as described above, 5% tails from the frequency distributionwere excluded and the 48 highest and 48 lowest plants from the remainingpopulation were selected for advancement to the next generation. Plantswithin each group were intermated as described above to obtain P₂progeny.

Histograms of the high and low myrosinase-selected P₂ populations areshown in FIG. 5. The results indicate that there were statisticallysignificant differences between high and low populations after twogenerations of selection (ANOVA:r² =18.3%; F=22.12; df=1,4; P=0.009).High and low populations differed by about two standard deviations inthe myrosinase selection experiment. The high population would beexpected to differ from the base population by about one standarddeviation, assuming that the mean of the base population lies halfwaybetween the means of the high and the low population. Statisticallysignificant heritabilities were calculated from parent-offspringregressions (h² =2 slope! for half sibs) (Myrosinase: h² =0.35; F=20.95;df=1,308; P<0.001).

A field experiment with the high and low myrosinase-selected populationswas carried out at the same time and at the same site as the fieldexperiment of Example 1. genotypes were planted in the field in arandomized design, and scored for insect herbivory. P₂ seeds wereplanted in the same split plot design as described in Example 1. Lack ofgermination or seed shortages reduced the number of experimental seedsby approximately 12 seeds per block. These effects were approximatelyequal between high and low selected lines. For the 10 blocks planted ineach experiment, there were about 840 experimental seeds planted in themyrosinase experiment.

Herbivory and fitness were measured as described in Example 1. Data wereanalyzed as described in Example 1. The herbivory data are summarized inTable 5.

                  TABLE 5                                                         ______________________________________                                        Herbivory by Flea Beetles on Myrosinase Populations                           Myrosinase    Mean Pit                                                        Population    Number                                                          ______________________________________                                        High          0.69 +/- 0.02.sup.a,*                                           Low           0.78 +/- 0.02.sup.a,*                                           ______________________________________                                         .sup.a (+/- 1 Standard Error)                                                 *Statistically significant, P = 0.043                                    

In the field, the population selected for increased myrosinase activitywas 10.3% more resistant than the low myrosinase line to flea beetles(FIG. 6, Table 5). This difference in resistance to flea beetles betweenhigh and low populations was statistically significant (P=0.043; Table6), and was only found to be significant for feeding on true leaves.

Plant fitness was 15.5% lower in the population selected for highmyrosinase concentrations (FIG. 6). This difference in the femalecomponent of fitness was statistically significant (P=0.016; Table 6).

Resistance to specialized lepidopteran larvae was not affected byselection for altered myrosinase activity. Instead, added nitrogenincreased herbivory by 59% compared to the ambient nitrogen levels(P=0.011; Table 6). No interaction between nitrogen and selectiontreatment was detected.

                  TABLE 6                                                         ______________________________________                                        ANOVA tables from split-plot field experiments for                            myrosinase in which high and low selected populations were                    compared for resistance and fitness parameters. Block {N}                     is the variation among blocks nested within the nitrogen (N)                  factor; Selection (S) is the variation between high and low                   selected populations; size is the covariate, cotyledon                        width.                                                                        F ratios.sup.a                                                                                 Cotyledon                                                                              Leaf   Leaf                                                          resistance                                                                             resistance                                                                           resistance                                                    to flea  to flea                                                                              to Lep.                                      Source   df      beetles  beetles                                                                              larvae Fitness                               ______________________________________                                        Nitrogen 1       3.97     0.33   11.02**                                                                              0.19                                  Block {N}                                                                              8       30.27*** 2.41*  6.80   13.31***                              Selection (S)                                                                          1       0.56     5.76*  1.67   9.22*                                 N*S      1       0.23     0.01   0.03   0.44                                  S*Block {N}                                                                            8       1.39     0.84   1.82   0.75                                  size     1       1.94     35.88***                                                                             25.52***                                                                             151.40***                             ERROR     527.sup.s                                                                            0.04     0.14   0.764  0.44                                  ______________________________________                                         .sup.a MS for F ratios and corresponding df in splitplot design as            specified in Steel, R. and Torrie, J., supra.                                 *P<0.05; **P<0.01; ***P<0.001                                                 .sup.s df for ERROR ranged from 527 to 747                               

Example 3 Selection for altered glucosinolate levels and myrosinaseactivity in Arabidopsis thaliana

Phenotypic analyses of total non-seed glucosinolate levels, myrosinaseactivity and insect leaf damage were carried out on homozygousrecombinant inbred lines of Arabidopsis thaliana. The lines were derivedfrom a cross between ecotypes Columbia and Landsberg erecta. Lister, C.and Dean, C., Plant J. 4:745-750 (1993). The lines were obtained fromthe Arabidopsis Biological Resource Center (Columbus, Ohio). Thirty-ninelines were grown in a randomized complete block design with 80 plantsper flat in 4 flats. Three weeks after planting, glucosinolateconcentrations were measured on a true leaf from each plant as describedin Example 1. Leaf areas were determined by computer scanning asdescribed in Example 1. Specific total non-seed glucosinolate levelswere calculated in units proportional to glucosinolate level per mm²leaf area. The mean specific glucosinolate level for each of 38recombinant inbred lines is shown in Table 7.

                  TABLE 7                                                         ______________________________________                                        Glucosinolate Levels and Myrosinase Activity                                  for Arabidopsis Recombinant Inbreds.                                          RI LINE        ADJ GS  Myrosinase                                             ______________________________________                                        5              0.769   0.772                                                  13             -0.390  -0.387                                                 14             0.672   -1.109                                                 17             0.379   -0.800                                                 25             -0.220  -0.317                                                 30             -0.820  0.319                                                  32             -0.398  -0.249                                                 33             0.643   -0.946                                                 34             -0.263  -0.153                                                 46             0.169   -0.188                                                 62             0.352   0.090                                                  67             0.275   0.688                                                  79             -0.011  0.175                                                  90             0.292   -0.567                                                 107            0.038   -0.413                                                 113            0.353   -1.099                                                 115            0.247   -0.546                                                 160            -0.625  -0.195                                                 167            -0.334  -0.277                                                 180            -0.130  -0.069                                                 194            -0.601  -0.773                                                 232            N/D     0.260                                                  235            0.280   1.176                                                  238            0.040   1.013                                                  263            -0.078  1.132                                                  264            -0.788  1.432                                                  288            0.153   0.214                                                  295            0.176   -0.122                                                 296            0.465   -0.250                                                 342            0.623   -0.086                                                 345            -0.709  -0.341                                                 350            -0.539  0.229                                                  358            -0.308  1.568                                                  363            -0.065  -0.062                                                 370            0.281   -0.352                                                 377            -0.625  0.162                                                  378            -0.387  1.821                                                  386            0.486   -0.817                                                 390            -0.702  0.193                                                  ______________________________________                                    

Myrosinase activity was determined as described in Example 2 on a leaftaken 3 weeks after planting, except that 40 minutes after the enzymereaction was initiated, A₄₉₀ measurements were taken at 2 minuteintervals until the completion of the 60 minute incubation. The velocityof the reaction was determined from the A₄₉₀ measurements and specificmyrosinase activity was calculated in units proportional to myrosinaseactivity per mm² leaf area. The mean specific myrosinase activity foreach of the 39 recombinant inbred lines is shown in Table 7.

One day after removing a leaf for glucosinolate and myrosinase analysis,a single first instar diamondback moth larvae was placed on a leaf ofeach plant. Leaf tissue consumed (mine size) was scored the followingday as described in Example 1, except that a scale of 0 to 3 was used.The data from Table 7 and the mean mine size for each recombinant inbredline were analyzed using quadratic least squares regression to estimatecovariant values for the three measurements; the regression analysis isplotted in FIG. 7.

A statistical analysis of the data in Table 7 is presented in Table 8.The results indicate that selecting for increased total non-seedglucosinolate levels and increased myrosinase activity reduces leafdamage by diamond back moth larvae.

                  TABLE 8                                                         ______________________________________                                        Effects of Glucosinolates and Myrosinase on                                   Diamondback Moth Larval Feeding                                               DEP VAR: MINE.sup.a                                                           SOURCE      DF    MEAN-SQUARE   F-RATIO                                                                              P                                      ______________________________________                                        SpGluc.sup.a                                                                              2     1.062         4.547  0.027                                  SpMyr.sup.a 2     1.156         4.948  0.021                                  SpGluc*SpMyr                                                                              1     1.768         7.568  0.014                                  LeafLen.sup.a                                                                             2     1.392         5.960  0.012                                  RI Line     39    0.324         1.389  0.243                                  ERROR       16    0.234                                                       ______________________________________                                         N = 63, R.sup.2 = 80.9%                                                       .sup.a Mine = Insect Score; SpGluc = Areaspecific glucosinolate level;        SpMyr = Areaspecific myrosinase activity; LeafLen = Leaf Length          

Example 4 Marker-assisted selection of glucosinolate and myrosinaselevels

RFLP linkage data for 101 Arabidopsis thaliana recombinant inbred (RI)lines, including the 39 lines analyzed for total glucosinolates,myrosinase and herbivory, is shown in Table 9. The data in Table 9 showinformation for 62 polymorphic markers, including chromosome, maplocation from an arbitrary starting point on the chromosome (incentiMorgans) and the polymorphic allele present in each RI line. Thedata are available from the Arabidopsis Biological Resource Center(Columbus, Ohio) and can be downloaded from the AAtDB database on theInternet (http://weeds.mgh.harvard.edu; electronic mailcurator@weeds.mgh.harvard.edu).

The marker data from Table 9 and the phenotypic data from Table 7 wereanalyzed by least squares interval mapping to identify polymorphicmolecular markers that were linked to altered myrosinase activity ortotal non-seed glucosinolate level (Haley, C. S. and Knott, S. A.,Heredity 69:315-324 (1992).

One genomic probe, cosmid m105, was found to be linked to altered levelsof myrosinase (Table 10). The genomic fragment present in cosmid m105 islocated on chromosome 3, approximately 25 map units from the arbitraryzero position at lambda clone m583. Two probes, cosmids g6842 andpCITd23, were found to be linked to altered levels of total non-seedglucosinolates (Table 10). The genomic fragment in cosmid g6842 maps tochromosome 2, approximately 29.2 map units from the zero position atm246. The fragment in pCITd23 maps to chromosome 4, approximately 18 mapunits from marker g3843.

TABLE 9

The following table contains genetic marker information from theColumbia×Landsberg recombinant inbred lines. A subset of these lines(numbers 5, 13, 14, 17, 25, 30, 32, 33, 34, 46, 62, 67, 79, 90, 107,113, 115, 160, 167, 180, 194, 232, 235, 238, 263, 264, 288, 295, 296,342, 345, 350, 358, 363, 370, 377, 378, 386, and 390) were used to mapmyrosinase activity and glucosinolate concentration quantitative traitloci in this cross, with the least squares interval mapping protocol ofHqaley and Knott (1992). The first paragraph (below) contains theidentification number for each recombinant inbred line, in the sameorder as subsequent paragraphs. Each subsequent paragraph contains thefollowing information; first, the name of the genetic marker, endingwith ab asterisk. Second, the chromosome containing this marker(numbered 1 to 5). Third, the location of the genetic marker incentiMorgans. Lastly, the allele for each recombinant inbred line, with"L" indicating the Landsberg allele, "C" indicating the Columbia allele,and "U" indicating unknown.

                                      TABLE 9                                     __________________________________________________________________________    4 5  13  14  17  519 619 25  29  30  32  33  34  35  36  37  46  52  53         54                                                                            59  62  67  68  71  79  84  90  107 113 115 123 125 131 160 161 166 167       173                                                                           175 177 179 180 181 182 188 190 191 193 194 199 209 214 217 231 232 235       237                                                                           238 240 242 245 253 257 259 263 264 266 267 279 283 284 288 295 296 297       302                                                                           303 311 321 332 342 345 349 350 351 356 358 359 363 367 370 377 378 386       390                                                                           394 395 397 398 400                                                         m488* 1 51                                                                             L C C L L C U L C C C L C L L C                                      H  L  C  C C C L L L C C C L L L C C L L                                      C  C  L  L C L L C L C C L L C C U L C L                                      C  C  C  C C L L L C L L L L L L C L C L                                      C  C  C  C L L L L L L C C L L L C L L L                                      L  C  C  L C C C C C                                                          g4715a* 1 55.4                                                                           L C C L L C U L C C C L C L L                                      C  U  L  C C C C L L U C C C L L L C C L                                      L  C  C  L L C L L C L C C L L C C U L C                                      L  C  C  C C C L L L C L L L L L L C L C                                      L  C  C  C C L L L L L L C C L U L C L L                                      L  L  C  C L C C C C C                                                        g3786* 1 64.5                                                                            L L L L L C U L C L C L C L C                                      C  L  L  L C C C L L L C C C L L L C C L                                      L  C  C  L L U L L C L C C L L C C U L C                                      L  C  U  C C L L L L C L L L L C L C L C                                      C  C  C  L C L L L C C L U C L U C C L L                                      L  U  C  C L C C C C C                                                        m235* 1 68.5                                                                             L L L L L C U L C L C L C L C                                      C  C  L  L C L C C L L C C C L L C C C C                                      L  C  C  L L L L C C L L C L C L C U L C                                      L  L  L  C C L L L L C L C L L C L C L C                                      C  C  C  L C L L L C C L L C L L C C C L                                      L  L  C  C L C C L L L                                                        g3829* 1 72.5                                                                            L L L L L C U L C L C L C L C                                      C  C  L  U C L C L L U C C U L L C C C C                                      L  C  C  L L L L C C L C C L C L C U L L                                      L  L  L  C C L L L L C L L L L U U C L C                                      C  C  C  C C L L L C C L U C L U C C C U                                      U  L  C  C L C U U L C                                                        m253* 1 82.5                                                                             C L L C L C U L L L L L L L C                                      C  C  L  L C C C C L L C C C L L C C C C                                      L  C  C  C C L C C C L L C L C C L U L C                                      L  L  L  C C L L L L C L L C L C C L L C                                      C  C  C  C C L L L L C L L C L L C L C L                                      H  C  C  C L C L L L L                                                        GapB* 1 92.5                                                                             U C L C U C U L U U L C L U C                                      C  U  L  U C C C C L U C C U L L C U U U                                      L  C  C  C C U U U C U L C L C C L U C L                                      L  L  U  U U L C C U U L L L U C U U U U                                      C  U  U  U C L L C C C U U C U U C U L U                                      C  C  C  U C C U U U L                                                        g4026* 1 110.4                                                                           C C C C L L L L C C L C C C C                                      C  C  L  L C C C C L L C C U C C L C L L                                      C  C  C  C L L C C C L C C L L C L C C C                                      L  C  C  C L C C C C C L L C L U L C C L                                      C  C  L  C C L C C C C C C L C L C L C C                                      C  C  C  C C C L L H L                                                        m315* 1 117.4                                                                            C C C C L L C L C C L C C C C                                      C  C  L  L L C C C L L C C L C C C C L L                                      C  C  C  C L C C C C C C C L L C L C C C                                      L  C  C  C L L C C C C C C C C C L C C L                                      C  L  L  C C L C C C C C C L L C L L C L                                      C  C  C  C C C C L C L                                                        g4552* 1 119                                                                           C C C C C L C L C C L C C C C C                                      C  C  L  L C C C L L C C U C C L U L L C                                      C  C  C  L C C C C C C C L L C L C C C C                                      C  C  C  L L C C C C C C C C U C C C C C                                      L  L  C  C L L C C C C C L L C L C C L L                                      L  C  L  C C C L C L                                                          m532* 1 135.3                                                                            L C C C C L U L C L C L C C L                                      C  C  C  L L L C C C C C C L C C C C L L                                      C  C  C  C L U C C C L C C L C C L U C L                                      C  C  U  C L C C C C C C C L C L C C C C                                      C  L  L  C C L L C C C C C C L C L L L L                                      L  L  L  C C C C C L L                                                        g17311* 1 141                                                                          L C C C C L U L C L C L C C L C                                      C  C  L  L L C C C C C C L C C C C L L C                                      C  C  C  L U C C L L C C C C C L U C L C                                      C  U  C  C L C C C L L C L C L C C C C L                                      L  L  L  L L L C L C C C C L C L L L L L                                      L  L  C  C C C C L L                                                          m246* 2 11.1                                                                             L C C C C L U L L L L L L L L                                      C  C  L  C L L L C L L C L L L L L C L C                                      C  L  C  C L L L C L L L L C C L C U L L                                      C  L  L  C L L L L L C C C C L L L L L C                                      L  L  L  L C C L L C L C C L L C L C L L                                      H  L  L  C C L C L L C                                                        g4133* 2 16.2                                                                            L C C C C L C L L C L L L L L                                      C  C  L  C L L L L L L L U U L L C L L C                                      C  L  L  C L L L C L L L L C C L C U L L                                      C  L  L  C L L L L L C C C L L L L L L L                                      L  L  L  C L C L L C L L C L L C C C L L                                      H  L  C  C C L C U C C                                                        g4532* 2 18.5                                                                            L C C C C C C L L C L L L L L                                      C  C  L  U L L L L L L L U U L L L L L C                                      C  L  C  C L L L C L L L L C C L C U L L                                      C  L  L  C L L L L L C C C C L U U L L L                                      L  L  L  C L C L L C L L C L L C L C L L                                      H  L  L  C C L C U L C                                                        g4553* 2 20.4                                                                            L C U C C L C L L L L L L L L                                      C  C  L  C L L L L L L C L L L L L C L C                                      C  L  C  C L L L C L L L L L C L C L L L                                      C  L  L  C L L L L U U C C C L U L L L C                                      L  L  L  C C C L L C L L C L L C L C L L                                      H  L  L  C C L C U L C                                                        m251* 2 47.7                                                                             L C C C C L U L L C L C L L L                                      L  C  L  C L C C L L L C L L L L C L L C                                      C  L  L  L L U L C L L L H C C L L U L L                                      L  C  U  C L L L L L C C C L L L L L L L                                      L  L  L  C L C C L C C L C L L L C L L L                                      C  L  L  C C L C L C L                                                        er* 2 56.5                                                                             L C C C C U U L L C L C L L C L                                      C  L  U  L U C L U L C C C L L C L L C L                                      L  L  L  L L L L L U L C C L L C C L L L                                      C  C  C  L L L L L L U U L L L L L U L L                                      C  L  C  L L L L C C L U L L L L U L L C                                      L  L  C  C L L L C L                                                          g6842* 2 58.5                                                                            L C C C C L U L L C L C L L C                                      L  C  L  C L C C L L U C C C L L C L L C                                      L  L  L  L L U C L L L L H C L L L U L L                                      L  C  U  C L L L L L L C C L L L L L L L                                      L  L  L  C L C L L C C L C L L U L L L L                                      C  L  L  C C L L U C L                                                        m323* 2 63.2                                                                             L C C C C L U L L C L C L C C                                      C  C  L  L L C C L C L C C L L C C L L C                                      L  L  L  L L U L L L L L C L L L L U C L                                      L  L  U  C L L L C L L C C L L U U L C L                                      L  C  L  C L L L L L C L C L C L C L L C                                      L  L  L  L C L L L C C                                                        m220* 2 64.5                                                                             L C C C C L U L L C L C L C C                                      L  C  L  C L C C L C L C C C L C C L L C                                      L  L  L  L L U L L L L L C C L L C U C L                                      L  L  U  C L L L C L L C C L L C L L C L                                      L  C  L  C L L L L C C L C L L L C L L L                                      C  L  L  L C L L L C C                                                        g17288* 2 71                                                                           L C C C C L C L L C L C L C C C                                      C  L  L  L C C L C L C C L L C C L L C L                                      L  L  L  L L L L L L L C L L L L C C L L                                      L  C  C  L L L C L L C C L L L L L C L L                                      C  L  C  L L L L L C L C L C L C L L C L                                      L  L  L  C L L L C C                                                          g4514* 2 76.4                                                                            L C C C C L L L L C L C L C C                                      C  C  H  L L L C L L L C U L L C C C L C                                      L  L  L  L L I L                                                                             L L L L C L L L L I C                                                                             L                                          L  L  U  L L C C C C L C C L L L C L C L                                      L  C  L  C L L L L L L L C L C L C L L C                                      L  L  L  L C L L L C C                                                        m336* 2 81.5                                                                             L C U C C L L L L C C C L C C                                      C  C  L  L L L C C C U C C L L C C C L C                                      L  L  L  C L L L L L L L C L L L L C C L                                      L  L  C  C L C C C C L C C L L L C L C L                                      L  C  L  C L L L L L L L C L C C C L L C                                      L  L  L  L L L L L C C                                                        m583* 3 7.7                                                                            L C C C L H U C C C L L L C L L                                      L  C  C  C C L C L L C L L C L C L L L L                                      C  L  L  C L C C C L L C C L C L U C C L                                      C  L  C  C L C L C C C C C L C L C C L C                                      L  C  C  L L C L C C L C L C U L L L C L                                      C  C  L  C L L C C C                                                          g4523* 3 11.8                                                                            L C C C L C U C C C L L L C L                                      L  L  C  C C C L C L U C L U C L C L L L                                      L  C  L  L C L C C C L L C C L C L U C C                                      L  C  L  C C L C L C C C C C L C L C C L                                      C  L  C  C L U C L C C L C L C U L L L C                                      L  C  C  L C L L L C C                                                        m228* 3 17.5                                                                             L C C C U L C C C C L C L C L                                      L  L  U  C C L L C L U C L U C L C L L L                                      L  C  L  L C L L C C L L C C L H L L C C                                      L  C  L  C C C C L L C C C C C U U L C L                                      C  C  C  C L C C L C C L C L C U C U L C                                      L  C  C  L C L L U C C                                                        g4708* 3 20.9                                                                            L C C C L L U C C C L C L C L                                      L  L  C  U C L L C L U C L L C L C L L L                                      L  C  L  L C L L C C L L C C L L L U C C                                      L  C  L  C C C C L L C C C C C C L L C L                                      C  C  C  C L C C L C C L C L C U C L L C                                      L  C  C  L C L L L C C                                                        m105* 3 24.7                                                                             L C C L L L U C C C L C L C L                                      L  L  C  C C L L C L L C L L C L C L L L                                      L  C  L  L C L L C C C L C C C H L U C L                                      L  C  L  C C C C L L C C C C C C L L C L                                      C  C  C  C L C C C C C L C L C C C L C C                                      L  L  C  L C L L L C C                                                        g4117* 3 61.5                                                                            C L C C L L U C C C L L L C C                                      L  C  C  C C C L C L L C C L C L C L L C                                      L  C  L  L L L L C C C C C L L L L U C L                                      C  L  L  C C C C L L C L C L C L L L C C                                      L  L  C  C C C C C L C C L C L L C L C L                                      C  L  C  L C L L L C C                                                        m249* 3 63.7                                                                             C L C C L L U C C C L L L C C                                      L  C  C  C C C L C L U C C L C L C L L C                                      L  C  L  L L L L C C C C C L L L L U C L                                      C  L  L  C C C C L L C L C L C L L L C C                                      L  L  C  C C C C C L C C L C L L C L C L                                      C  L  C  L C L L L C C                                                        g4564b* 3 63.9                                                                           C L C L L L U C C C L L L C C                                      L  C  C  C C C C C L U C C U C L C L L C                                      L  C  L  L L L L C C C C L L L U L U C L                                      C  L  L  C C C C L C C L C L C U L L L C                                      L  L  C  C L C C C L C C U C L U C L C L                                      C  L  C  L C L U U C C                                                        m457* 3 71.9                                                                             C L L L L L U C L C L L L C C                                      C  C  C  L C C C C L L C L L C L C L L C                                      L  C  L  L L L L C C C C L L L L L U C C                                      C  L  L  C C C L L C C L C L L C C L L C                                      L  L  L  L C C C C L C C L C L L C L C L                                      C  L  C  L C L L L C C                                                        g4014* 3 72                                                                            C L U U U L C C L C L L L C C C                                      C  C  C  C C C C L L C C U C L C L L C L                                      C  L  L  U L L C C C C L U U L U L C C C                                      L  L  C  C C C L C C L C L L U U L L C L                                      L  C  C  L C C C L C C L C L L C L C L C                                      L  C  L  C U L U C C                                                          g2778* 3 86.3                                                                            C L L L C L U C L C L L L C C                                      C  C  C  L L C C C L U C L U C L C L L C                                      L  C  L  C L U L C C C C C L L L C U C C                                      C  L  U  C C C L L C C L C C L L U L C C                                      L  L  L  C C C C C L C C U C L U C L C L                                      C  L  C  L C L L L C C                                                        m424* 3 91.3                                                                             C L C L C L U L L C L C L C C                                      C  C  C  L L C C L L U C L L C L L L L C                                      L  C  L  C L U L C C C C C L C L C U C C                                      C  L  U  C C C L L C C L C C L L C L C L                                      L  L  L  C C C C C L C C L C L L C L C L                                      C  L  C  L C L L L C C                                                        g3843* 4 4                                                                             C L L C C L U C L L L C L C C C                                      L  C  L  C C C C L U L L C L C L L L C L                                      L  C  L  C C C L L C L L C L L L U C C L                                      L  C  L  L C L C L L L L C L L L L C C L                                      L  C  C  L L L C C C C U L L L L L L L L                                      C  C  L  L L C C L L                                                          g2616* 4 4                                                                             C L L C C L U C L C L L L C C C                                      L  L  L  C C C C L L L L C L C L L L C L                                      L  C  L  C U C C L C L L C L L L U C C L                                      L  U  L  L C L C C L L L C L L L L C C L                                      L  C  C  L L L L C C L U L L L C L L L L                                      C  C  L  L L C C L L                                                          m506* 4 14.9                                                                             C L L C C L U C L C L L L C C                                      C  L  L  L C C C C L L L L C L C L L L C                                      L  L  C  L C U C C L C L L C L L L U C C                                      L  L  U  L L C L C C L L L C L L L L C C                                      L  L  C  C L L L L C C L L L L L C L L L                                      L  C  C  L L L C C L L                                                        m518* 4 30                                                                             C C U C C L U C C C L L L L C L                                      L  L  L  C C C C L L L L C C C L C L C L                                      L  C  L  C L C C L C L C C L L L U C C L                                      L  L  L  L C L C C L L L L L C L L L L L                                      L  C  C  L L L L C C L L L L L C C L L L                                      C  C  C  L C C C L C                                                          pCITd23* 4 31.2                                                                          C C L C C L C C C C L L L L C                                      L  L  C  L C C C C L L L L C C C L C L C                                      L  L  C  L C L C C L C L C C L L L C C C                                      L  L  C  L L C L C C L L L L L L L L L L                                      L  C  C  C L L L L C C L L L L L C C L L                                      L  L  C  C L C C C L L                                                        g6837* 4 39                                                                            C C L C C C C C C C L L L L C L                                      L  C  U  C C C C L L L L C C C C C L C L                                      C  C  C  C L C C L C C C C C L C C C C L                                      C  C  L  L C L L C L L L L L L L C L L L                                      C  L  C  L L L L C C L L C L L C C C L H                                      L  C  C  L C C C C L                                                          m326* 4 39.2                                                                             C C L C C C C C C C L L L L C                                      L  L  C  L C C C C L L L L C C C C C C C                                      L  C  C  C C L C C L L C C C C L C C C C                                      L  C  C  L L C L L C L L L L L L L C L C                                      C  C  L  C L L L L C C L L C L L C C C L                                      H  L  C  C L C C C C L                                                        g10086* 4 42.2                                                                           C C L C C U C C C C L L L L C                                      L  L  C  L C C C C L L L L C C C C C L C                                      L  C  C  C C L C C L C C C C C L C C C C                                      L  C  C  L L C U L C L L U L L L L C L C                                      C  C  L  C L L L L C C L L C L L C C C L                                      H  L  C  C L C C C C L                                                        m226* 4 43.7                                                                             C C L C C C C C U C L L C L C                                      L  L  L  L C C C C L L L L C C C C C C C                                      L  C  C  C C L C C C L C C C C L C C C L                                      L  C  C  L L C L L C L L L L L L L C L H                                      C  C  L  C L L L L C L L L C L L C C C L                                      U  L  C  C L C C C C C                                                        g4564a* 4 44                                                                           C C L C C C U C C C L L L L C L                                      L  C  L  C C C C L L L L C C C C C L C L                                      C  C  C  C L C C L C C C C C L C C C C L                                      C  C  L  L C L L C L L L L L L L C L C C                                      C  L  C  L L L L C C L L C L L C C C L H                                      L  C  C  L C C C C L                                                          g3845* 4 51                                                                            C C L C C C C C C C L L L L C L                                      L  L  U  C C C C L L L L U C C C C C C L                                      C  C  C  C L C C C L C C C C L C C C L L                                      C  C  L  L C L L C L L L L L L L C L H C                                      C  L  C  L L L L C L L U C L U C C C L C                                      L  C  C  L C C C C C                                                          m600* 4 63.5                                                                             C C C C U L U C C L L L C C C                                      L  L  U  L C L L C L U C L U C C C U L C                                      L  C  C  C L U C C C L C L C C L C C L L                                      L  C  C  L L C L L C L L L L L U U C L H                                      C  C  L  C L C L L C L L L C L U C U C L                                      C  L  C  C C C C U C L                                                        g8300* 4 63.9                                                                            C C C C C L U C C L L L C C C                                      L  L  L  L C U L C L U C L C C C C L L C                                      L  C  C  C L U C C C L C L C C C C U L L                                      L  C  U  C L L L L C C L L L L C C C L C                                      C  C  L  C C C L L C L L L C L U C C C L                                      C  L  C  C C L C C C L                                                        pCITd99* 4 68                                                                          L C C C U L L C C L L L C C C L                                      L  L  L  C U L C L U C L U C C C L C C L                                      C  C  L  L U C L C L C L L C C C C L L L                                      C  L  L  U L L L C C L L L L U U C L U C                                      C  L  C  L C L L L L L L C L U C U C L C                                      L  C  C  C L C U C L                                                          g3088* 4 70.4                                                                            C C C C C L U C C L L L C C C                                      L  L  L  U C U L C L U C L C C C C L L C                                      L  C  C  L L L C L C L C L C C C C U L L                                      L  C  L  L H L L L C C L L L L C C C L C                                      C  C  L  C C C L L C L L U C L U C C C L                                      C  L  C  C C L C C C L                                                        g3713* 4 85.4                                                                            L C L C C C U C C L L L C C L                                      L  L  L  L L C L C C U L L C C C C L C L                                      L  C  L  C L L C L C L C C L C C C U L L                                      C  C  L  L C L L L L C L L L L C C L L C                                      C  C  L  L L C L L L C L U C L C C C C L                                      C  L  C  C C L C C L L                                                        g3715* 5 8.4                                                                           C C L C L L U L L C L C C L L C                                      L  L  L  C L L C L C L L C L C L L L L L                                      L  L  L  L C C L L L L L L L C L U L L C                                      L  C  L  C C L L L L L L C L L C C C L L                                      C  L  L  C L L C C L L U C L C C C C L L                                      C  L  C  L C L C L L                                                          m217* 5 10.6                                                                             C C L L L L U L L C L C C L L                                      C  C  L  L C L L C L C L L C L C L L L L                                      L  L  L  L L C C L L L L L L L C C U L L                                      C  C  C  L L C L L L L L L C L C C C L L                                      L  C  L  L C L L C C L L C C L C C C C L                                      L  C  L  C L C L C C L                                                        g3837* 5 14.5                                                                            C C L L L L U L L C L C C L L                                      C  C  L  L C C L C L C L L C L C L L L L                                      L  L  L  L L C C L L L L L L L C C U L L                                      C  L  C  L L C L L L L L L C L C C C L L                                      L  C  L  L C L L C C L L U C L C C C C L                                      L  C  L  C L C L C C L                                                        g4560* 5 27.9                                                                            C C C L L L U L L L L C C C L                                      C  C  C  L C C L C L C L L C L L L L L L                                      L  L  L  L L U L L C L L L C C C C U L L                                      L  L  U  L L C L L U L L L L L C C C L C                                      L  L  L  L L L L C L L L C C L L C L C U                                      U  L  L  C L C C C C L                                                        m291* 5 34.4                                                                             C L C L L C C L L L L C C C L                                      C  C  C  L C C L C L C L L L L L L L L L                                      L  L  L  L U L L L C L L L C C C C L L L                                      L  L  L  L C C L L L L L L L C C C C L C                                      L  L  L  L U U U U L L L C C L L U L U L                                      L  L  L  U U U C C C L                                                        m247* 5 52.5                                                                             C C C C L L U L C L L C C C L                                      C  L  C  L C C C C L L L L L L L C C L C                                      L  C  C  L C C L C C L L L C L C L U C L                                      C  C  C  C C C L L C L C L C L C C C L L                                      L  L  L  L L L L C L L L C C C L C L C C                                      L  L  C  L L C L C C C                                                        g4028* 5 57.7                                                                            L C C C L L U L C L L C C C L                                      C  L  C  L L C C C L L L L L L L C C L C                                      L  C  C  L L C L C C L L L C L C L U L L                                      L  C  C  C C C L L C C C L C L C C C L L                                      C  L  L  L L C L C L L L C C C L C L C C                                      L  L  H  L L C L C C C                                                        m435* 5 80.1                                                                             L C L L C L U C L C L C L L L                                      L  L  C  L L C C C L L C L L L C C L L C                                      L  C  C  L L C C L C L L L C L C L U C L                                      L  C  C  C C C L C L C C L C L C L C L C                                      C  C  L  L C C C L C L L C C C L C L C C                                      L  C  C  L L C C C C C                                                        g2368* 5 93.5                                                                            L L L L C L U C L C L L L L L                                      L  C  C  L L C L C L L C L L L C C L L C                                      C  C  C  L L C C L C L C L C L C L U C L                                      C  C  C  C C L L C L L C L C L C U C L C                                      C  C  L  L C C C L C L L U C C C C C C C                                      C  C  C  L C C C C C C                                                        m555* 5 100                                                                            L U L L U L C C C C L L L L L C                                      C  C  L  L C L C L U C L L L C C L L C C                                      C  C  L  L C C C C C C L C L C L C C L C                                      C  C  C  C C L C L L C C C C U L C L C C                                      C  L  L  C C C L C L L C C C L C C C C C                                      C  L  L  C C C L C C                                                          __________________________________________________________________________

                  TABLE 10                                                        ______________________________________                                               SUM-OF-                                                                SOURCE SQUARES   DF     MEAN-SQUARE                                                                             F-RATIO P                                   ______________________________________                                        Myrosinase QTL: G29 = m 105 at III:25                                         DEP VAR: MYR N: 39 MULTIPLE R: 0.484                                          SQUARED MULTIPLE r: 0235                                                      G29    4.639     1      4.639     11.344  0.002                               ERROR  15.131    37     0.409                                                 Glucosinolate QTL: g19 = g6842 at II:59; g 41 = pCITd23 at IV:31              DEP VAR: GS N: 38 MULTIPLE R: 0.614                                           SQUARED MULTIPLE R: 0.377                                                     G19    1.269     1      1.269     9.060   0.005                               G41    1.158     1      1.158     8.270   0.007                               G19*G41                                                                              0.054     1      0.054     0.383   0.540                               ERROR  4.762     34     0.140                                                 ______________________________________                                    

These results show that marker-assisted selection can be used toidentify plant lines having reduced cotyledon or leaf feeding by insectpests of Brassicaceae.

Example 5 Selection for altered glucosinolate levels and myrosinaseactivity in Brassica napus

About 10,000 seeds of Brassica napus cultivar IMC-01 (IMC Cargill Foods,Wayzata, Minn.) are preimbibed in 300-seed lots for two hours on wetfilter paper to soften the seed coat. The preimbibed seeds aremutagenized by incubating in 80 mM ethyl methanesulfonate (EMS) for fourhours. Following mutagenesis, the seeds are rinsed three times indistilled water. The seeds are sown in 48-well flats containing Pro-Mix.About 68 percent of the mutagenized seed will germinate. The plants aremaintained at 25°/15°C, 14/10 hour day/night conditions in a greenhouse.At flowering, each plant is individually self-pollinated.

Seed (termed M₂ seed) from individual plants is individually cataloguedand stored. Approximately 5,000 M₂ lines are planted in a summernursery. The seed from each selfed plant is planted in 3 meter rows with6 inch row spacing. IMC-01 is planted as the check variety.

Ten cotyledons from M₂ seedling rows and IMC-01 control seedling rowsare each analyzed in bulk for total non-seed glucosinolate levels asdescribed in Example 1 and for myrosinase activity as described inExample 3. Statistical thresholds for glucosinolate levels andmyrosinase activity are established from the control IMC-01 cotyledonanalyses. Zar, J., supra, pp. 83-86. Those plants exceeding thestatistical thresholds for both increased total non-seed glucosinolatesand increased myrosinase activity are self-pollinated by bagging themain raceme of each plant. At maturity, selfed plants are individuallyharvested and M₄ seeds are catalogued and stored.

M₄ seed is planted in a greenhouse in 4 inch pots containing Pro-Mixsoil and the plants maintained at 25°/15° C, 14/10 hour day/night cycle.Cotyledons from M₄ seedlings and IMC-01 controls are individuallyanalyzed for glucosinolate levels and myrosinase activity as describedabove. Statistical thresholds are established from 250 IMC-01 controlseedlings. At flowering, the terminal raceme is self-pollinated bybagging. At maturity, M₅ seed from plants exceeding the statisticalthreshold for glucosinolate and myrosinase is individually harvestedfrom each M₄ plant, labeled and stored.

Selected M₅ seed is planted in a field trial in 3 meter rows with 6 inchrow spacing. Cotyledons from 10 M₅ seedlings in each row are analyzedfor glucosinolate and myrosinase as described above, using the sameZ-distribution as for M₄ population. Plants exceeding the statisticalthreshold are self-pollinated and the remaining open-pollinated plantsin the same row are bulk harvested.

Selected M₆ seed is entered into field trials at 4 locations in easternIdaho. The four locations are chosen to have differences among thelocations in growing conditions. The selected M₆ seed lines are plantedin four 3-meter rows with an 8-inch spacing. Each plot is replicatedfour times. The planting design is a randomized complete block. Thecultivars IMC-01, Westar, Global and Cyclone are used as checkcultivars. Cotyledons of 10 seedlings from each block are analyzed forglucosinolate and myrosinase levels as described above. Cotyledon andleaf damage by flea beetles and lepidopteran larvae are estimated asdescribe in Example 1. M₆ plants are also evaluated for agronomiccharacteristics such as yield, standability, disease resistance and seedcharacteristics such as low erucic acid and low seed glucosinolatelevels. The plots are harvested at maturity and the yield of each entryis determined by taking the statistical average of the fourreplications.

The Least Significant Difference test is used to rank the entries foryield, insect damage, total non-seed glucosinolate levels and myrosinaseactivity. M₆ lines show statistically significant increases in totalnon-seed glucosinolate levels and myrosinase activity, compared to checkvarieties. M₆ lines also show statistically significant reductions insusceptibility to insect damage compared to check varieties. Those M₆lines inheriting increased non-seed glucosinolate and myrosinaseactivities as well as other desired characteristics, are advanced tosubsequent field trials.

To the extent not already indicated, it will be understood by those ofordinary skill in the art that any one of the various specificembodiments herein described and illustrated may be further modified toincorporate features shown in other of the specific embodiments.

The foregoing detailed description has been provided for a betterunderstanding of the invention only and no unnecessary limitation shouldbe understood therefrom as some modifications will be apparent to thoseskilled in the art without deviating from the spirit and scope of theappended claims.

What is claimed is:
 1. A method for producing a cruciferous plant havinga heritable reduction in susceptibility to cotyledon or leaf feeding byPhyllotreta insects, comprising the steps of:a) selecting, in apopulation of P₀ Brassicaceae plants having a mean total non-seedglucosinolate level, at least one P₀ plant having a total non-seedglucosinolate level that is decreased sufficiently, relative to saidtotal non-seed glucosinolate level in said P₀ population, to reducesusceptibility to cotyledon or leaf feeding by Phyllotreta insects; b)producing P₁ progeny from said at least one P₀ plant; c) identifying atleast one P₁ plant that inherits said decreased total glucosinolatelevel, thereby producing said plant having said reduced susceptibilityto cotyledon or leaf feeding by said insects.
 2. The method of claim 1,wherein said P₁ progeny are produced by selfing.
 3. The method of claim1, wherein said selecting step comprises selecting a plurality of P₀plants having a total non-seed glucosinolate level that is decreasedsufficiently, relative to said total non-seed glucosinolate level insaid P₀ population, to reduce cotyledon or leaf feeding by Phyllotretainsects and wherein said P₁ progeny are produced by crosses among saidplurality of P₀ plants.
 4. The method of claim 1, wherein said P₀population comprises plants grown from mutagenized seeds.
 5. The methodof claim 1, wherein said at least one P₀ plant or said at least one P₁plant is identified by genetic linkage between said decreased totalnon-seed glucosinolate level and a polymorphic genetic marker.
 6. Themethod of claim 5, wherein said genetic marker comprises a nucleic acidhaving substantial sequence similarity to about 50 nucleotides fromArabidopsis RFLP probe g6842.
 7. The method of claim 5, wherein saidgenetic marker comprises a nucleic acid having substantial sequencesimilarity to about 50 nucleotides from Arabidopsis RFLP probe pCITd23.8. The method of claim 1, wherein said P₀ plant is selected from plantsin the 0-15 percentile for total non-seed glucosinolates in said P₀population.
 9. The method of claim 1, wherein said plant is a Brassicacampestris plant.
 10. The method of claim 1, wherein said plant is aBrassica napus plant.
 11. The method of claim 1, wherein saidPhyllotreta insects comprise Phyllotreta cruciferae.
 12. A method forproducing a cruciferous plant having a heritable reduction in cotyledonor leaf feeding by Phyllotreta insects, comprising the steps of:a)selecting, in a population of P₀ Brassicaceae plants having a meanmyrosinase activity, at least one P₀ plant having a level of myrosinaseactivity that is increased sufficiently, relative to said myrosinaseactivity in said P₀ population, to reduce susceptibility to cotyledon orleaf feeding by cruciferous insects; b) producing P₁ progeny from saidat least one P₀ plant; c) identifying at least one P₁ plant thatinherits said increased myrosinase activity, thereby producing saidplant having said reduced susceptibility to cotyledon or leaf feeding bysaid insects.
 13. The method of claim 12, wherein said P₁ progeny areproduced by selfing.
 14. The method of claim 12, wherein said selectingstep comprises selecting a plurality of P₀ plants having a myrosinaselevel that is increased sufficiently, relative to said myrosinaseactivity in said P₀ population, to reduce susceptibility to cotyledon orleaf feeding by Phyllotreta insects and wherein said P₁ progeny areproduced by crosses among said plurality of P₀ plants.
 15. The method ofclaim 12, wherein said P₀ population comprises plants grown frommutagenized seeds.
 16. The method of claim 12, wherein said P₀ plant isidentified by genetic linkage between said increased myrosinase activityand a polymorphic genetic marker.
 17. The method of claim 16, whereinsaid genetic marker comprises a nucleic acid having substantial sequencesimilarity to about 50 nucleotides from Arabidopsis RFLP probe m105. 18.The method of claim 12, wherein said P₀ plant is selected from plants inthe 85-100 percentile for myrosinase activity in said P₀ population. 19.The method of claim 12, wherein said plant is a Brassica campestrisplant.
 20. The method of claim 12, wherein said plant is a Brassicanapus plant.
 21. The method of claim 12, wherein said Phyllotretainsects comprise Phyllotreta cruciferae.
 22. The method of claim 12,wherein said at least one P₀ plant is further selected to have a levelof total non-seed glucosinolates that is increased sufficiently,relative to a mean total non-seed glucosinolate level in said P₀population, to reduce susceptibility to cotyledon or leaf feeding bycruciferous insects.
 23. A cruciferous plant produced by the method ofclaim
 1. 24. A cruciferous plant produced by the method of claim
 12. 25.A cruciferous plant produced by the method of claim 22.